Prenyl Pterocarpans from Algerian Bituminaria bituminosa and Their Effects on Neuroblastoma

The pterocarpan fraction from aerial parts of Bituminaria bituminosa was investigated for both chemical characterization and biological evaluation. Chemical studies were in accordance with the literature data on Bituminaria genus resulting in the identification of typical 4,8-prenyl pterocarpans. Three new members, bituminarins A–C (1–3), were isolated along with main bitucarpin A (4), erybraedin C (5) and erybraedin D (6) already reported from this plant. Further, biological studies evidenced antiproliferative properties of the most abundant pterocarpans 4 and 5 on neuroblastoma SH-SY5Y cell line, in agreement with previously described antiproliferative activity of these compounds against cancer cell lines other than neuroblastoma. The structure and the stereochemistry of the new molecules was determined by extensive spectroscopic analysis and chemical derivatization methods. The biological investigation was carried out by using an assay platform based on a live-cell imaging system revealing an apoptotic cell death induction.


Introduction
Bituminaria bituminosa (L.) Stirt.(syn.Psoralea bituminosa L.), commonly known as pitch trefoil, is a scrambling perennial legume species (Fam.Fabaceae) mainly distributed in Mediterranean coastal regions [1].The plant has pinkish-mauve flowers and trifoliate leaves with a characteristic smell of bitumen [1].B. bituminosa produces a large amount of biomass with significant nutritive value that is mainly utilized for feeding sheep and goats [2] even though some concern on the forage use has been raised due to the presence of photoreactive compounds in the plant secondary metabolite pool [3].The phytochemicals described in the literature for B. bituminosa include different classes of compounds, with pterocarpans and furanocoumarins being the most typical components of the aerial parts of the plant [4][5][6][7].Pterocarpans and furanocoumarins have also been detected in the volatile fraction from leaves and flowers [8].Among pterocarpans, bitucarpin A (4) and erybraedin C (5) are the most abundant compounds detected in the leaves of the plant [5,6,9].Other main phytochemicals reported from the aerial parts of B. bituminosa comprise caffeic and coumaric acids, apigenin glucosyl derivatives, luteolin, daidzein, lignans and soyasaponins [10,11].
Pterocarpans are potent plant defensive metabolites (phytoalexins), mainly produced by species belonging to Fabaceae family, that have been demonstrated to have a variety of interesting pharmacological properties [12].Naturally occurring pterocarpans are the second largest group of isoflavonoids [13] and are often used in traditional medicine in different countries as an alternative and supplementary therapy.Interestingly, literature data show that pterocarpans from Bituminaria species are characterized by the presence of one or two prenyl substituents, which are typically located at C-4 and C-8 on the benzofuran-benzopyran tetracyclic ring system of the pterocarpan nucleus [14].
In continuing our studies on Algerian plants used in traditional medicine [15][16][17], we examined the non-polar extract of aerial parts of B. bituminosa that was sampled in the region of Fessdis (Batna), in June 2015.The chemical study resulted in the characterization of the pterocarpan fraction including three unprecedented compounds, bituminarins A-C (1-3), co-occurring as minor metabolites along with main bitucarpin A (4) [4], erybraedin C (5) [18,19] and erybraedin D (6) [19,20] (Figure 1), already reported from B. bituminosa.Additional furanocoumarins, psoralen (7) [21][22][23] and isopsoralen [24] (8), and plicatin B (9) [25] (Figure 2) were also identified in the extract (see Section 4) in agreement with previous reports on Bituminaria chemistry.Bitucarpin A has been reported to have anticlastogenic activity in lymphocytes [26], whereas erybraedin C is known to exhibit cytotoxic effects [27] and inhibitory activity on human topoisomerase I [28].Due to their considerable pharmaceutic interest, additional biological properties of bitucarpin A (4) and erybraedin C (5) have been explored with regard to their putative antiproliferative activity against SH-SY5Y neuroblastoma cells.Pterocarpans are potent plant defensive metabolites (phytoalexins), mainly produced by species belonging to Fabaceae family, that have been demonstrated to have a variety of interesting pharmacological properties [12].Naturally occurring pterocarpans are the second largest group of isoflavonoids [13] and are often used in traditional medicine in different countries as an alternative and supplementary therapy.Interestingly, literature data show that pterocarpans from Bituminaria species are characterized by the presence of one or two prenyl substituents, which are typically located at C-4 and C-8 on the benzofuran-benzopyran tetracyclic ring system of the pterocarpan nucleus [14].
In continuing our studies on Algerian plants used in traditional medicine [15][16][17], we examined the non-polar extract of aerial parts of B. bituminosa that was sampled in the region of Fessdis (Batna), in June 2015.The chemical study resulted in the characterization of the pterocarpan fraction including three unprecedented compounds, bituminarins A-C (1-3), co-occurring as minor metabolites along with main bitucarpin A (4) [4], erybraedin C (5) [18,19] and erybraedin D (6) [19,20] (Figure 1), already reported from B. bituminosa.Additional furanocoumarins, psoralen (7) [21][22][23] and isopsoralen [24] (8), and plicatin B (9) [25] (Figure 2) were also identified in the extract (see Section 4) in agreement with previous reports on Bituminaria chemistry.Bitucarpin A has been reported to have anticlastogenic activity in lymphocytes [26], whereas erybraedin C is known to exhibit cytotoxic effects [27] and inhibitory activity on human topoisomerase I [28].Due to their considerable pharmaceutic interest, additional biological properties of bitucarpin A (4) and erybraedin C (5) have been explored with regard to their putative antiproliferative activity against SH-SY5Y neuroblastoma cells.Pterocarpans are potent plant defensive metabolites (phytoalexins), mainly produced by species belonging to Fabaceae family, that have been demonstrated to have a variety of interesting pharmacological properties [12].Naturally occurring pterocarpans are the second largest group of isoflavonoids [13] and are often used in traditional medicine in different countries as an alternative and supplementary therapy.Interestingly, literature data show that pterocarpans from Bituminaria species are characterized by the presence of one or two prenyl substituents, which are typically located at C-4 and C-8 on the benzofuran-benzopyran tetracyclic ring system of the pterocarpan nucleus [14].
The presence in the 13 C NMR spectrum of carbon resonances at δ C 78.8 (C, C-3 ′ ), 69.6 (CH, C-2 ′ ), and 27.1 (CH 2 , C-1 ′ ) that could be unequivocally attributed to the carbon atoms of a 3,3-dimethyl-2-hydroxy pyran ring [30] confirmed the structural hypothesis.The HMBC experiment was crucial to establish the position of both pyran ring and prenyl chain (Figure 3).Indeed, diagnostic long-range correlations were observed between H 2 -1 ′ (δ H 2.88 and 2.48) with C-4 (δ C 108.9) and C-4a (δ C 154.5) as well as between H 2 -1 ′′ (δ H 3.21 and 3.27) with C-7 (δ C 126.0), C-8 (δ C 120.7), and C-9 (δ C 155.9), thus allowing one to locate the pyran ring at C-3/C-4 and the prenyl group at C-8 of the pterocarpan scaffold.Other significant long-range correlations are reported in Figure 1.The coupling constant value (J = 6.9 Hz) between angular protons H-6a and H-11a, as well as their NOESY correlation, indicated a cis-junction between the chroman and benzofuran rings as reported for almost all natural pterocarpans.Moreover, the CD spectrum, which showed a positive curve at 286 nm, and the negative specific rotation [α] D −268.0 (c 0.013, CHCl 3 ), were consistent with a 6aR,11aR absolute configuration [31].The absolute configuration of the stereogenic center C-2 ′ was deduced to be R by applying the modified Mosher method (Figure 4).
the presence of a pterocarpan skeleton.
The presence in the 13 C NMR spectrum of carbon resonances at δC 78.8 (C, C-3′), 69.6 (CH, C-2′), and 27.1 (CH2, C-1′) that could be unequivocally attributed to the carbon atoms of a 3,3-dimethyl-2-hydroxy pyran ring [30] confirmed the structural hypothesis.The HMBC experiment was crucial to establish the position of both pyran ring and prenyl chain (Figure 3).Indeed, diagnostic long-range correlations were observed between H2-1′ (δH 2.88 and 2.48) with C-4 (δC 108.9) and C-4a (δC 154.5) as well as between H2-1″ (δH 3.21 and 3.27) with C-7 (δC 126.0),C-8 (δC 120.7), and C-9 (δC 155.9), thus allowing one to locate the pyran ring at C-3/C-4 and the prenyl group at C-8 of the pterocarpan scaffold.Other significant long-range correlations are reported in Figure 1.The coupling constant value (J = 6.9 Hz) between angular protons H-6a and H-11a, as well as their NOESY correlation, indicated a cis-junction between the chroman and benzofuran rings as reported for almost all natural pterocarpans.Moreover, the CD spectrum, which showed a positive curve at 286 nm, and the negative specific rotation [α]D −268.0 (c 0.013, CHCl3), were consistent with a 6aR,11aR absolute configuration [31].The absolute configuration of the stereogenic center C-2′ was deduced to be R by applying the modified Mosher method (Figure 4).Bituminarin B (2) exhibited strong similarities with bituminarin A (1).It had the sam molecular formula C25H28O5 as deduced by the peak at m/z 407.1878 in the HR-ESIMS spe trum (negative mode).Signals in the 1 H NMR and 13 C NMR spectra of 2 were almost supe imposable with those of 1 implying a strict structural correlation.Analysis of 2D NMR e periments of 2 clearly confirmed that the planar structure is the same as 1, indicating th the difference between two compounds should be ascribed to stereochemical features.T 6a,11a-cis-junction of 2 was assigned by the J6a,11a (Table 1) as well as NOE interactions an ogous to 1. Further, the CD profile and [α]D = -194.5(c 0.013, CHCl3) indicated the 6aR,11 absolute configuration also for bituminarin B (2) [31].Consequently, the difference had be in C-2′ configuration, opposite to that of 1.In fact, consistent with this, comparison 1 Bituminarin B (2) exhibited strong similarities with bituminarin A (1).It had the same molecular formula C 25 H 28 O 5 as deduced by the peak at m/z 407.1878 in the HR-ESIMS spectrum (negative mode).Signals in the 1 H NMR and 13 C NMR spectra of 2 were almost superimposable with those of 1 implying a strict structural correlation.Analysis of 2D NMR experiments of 2 clearly confirmed that the planar structure is the same as 1, indicating that the difference between two compounds should be ascribed to stereochemical features.The 6a,11a-cis-junction of 2 was assigned by the J 6a,11a (Table 1) as well as NOE interactions analogous to 1. Further, the CD profile and [α] D = −194.5(c 0.013, CHCl 3 ) indicated the 6aR,11aR absolute configuration also for bituminarin B (2) [31].Consequently, the difference had to be in C-2 ′ configuration, opposite to that of 1.In fact, consistent with this, comparison of proton spectra of 1 and 2 showed that the main difference was in the 1 H NMR pattern of H 2 -1 ′ methylene signals (Table 1).This difference was more evident by comparing the 1 H NMR spectra recorded in C 6 D 6 of the acetyl derivatives of 1 and 2, compounds 1a and 2a, respectively (see Section 4 and Supplementary Materials).In compound 1a, H 2 -1 ′ methylene resonated as a broad triplet at δ H 2.91 (2H, J = 4.8 Hz), whereas in compound 2a these proton signals were split into two double doublets at δ H 2.87 (1H, J = 17.7, 4.7 Hz) and 3.01 (1H, J = 17.7, 5.3 Hz) (Figure 5), in agreement with a different orientation of 2 ′ -OH group.Therefore, bituminarin B (2) was the 2 ′ -epimer of bituminarin A (1). Complete NMR assignment of 2 is reported in Table 1 (CD 3 COCD 3 ) and in C 6 D 6 (Section 4).
Molecules 2024, 29, 3678 The signal multiplicity of the methylene H2-1′ in compound 3a appeared si those of compound 2a, thus suggesting the same S configuration at C-2′, where figuration at C-2″ remained unassigned.

Biological Activity: Antiproliferative Activity of Bitucarpin A (4) and Erybraedin Neuroblastoma SH-SY5Y Cells
The biological properties of bitucarpin A (4) and erybraedin C (5) have b ated on neuroblastoma SH-SY5Y cells, that to the best of our knowledge have n tested with pterocarpan compounds.First, with the aim at establishing the rang tive concentrations for the in vitro studies, a cell growth dose-response bioassa us to determine the IC50 of both bitucarpin A and erybraedin C. For this pu growth percentage was measured by a live-cell scan assay performed through Incucyte system after 24 h of treatment with 4 and 5, both at log-fold concent tablishing 100% of the cell growth from the untreated cells (Figure 6).This e aided in establishing IC50 = 0.2048 µg/mL and IC50 = 0.1976 µg/mL for bitucar erybraedin C, respectively.Successively, the effects on ROS production in SH line induced by bitucarpin A (4) and erybraedin C (5), both tested at 1 µg/m µg/mL, were measured by CellRox assay in the same Zoom Incucyte live-cell sc Data were collected every two hours over 48 h of cell incubation by comparin time-matched cells incubated without pterocarpans (i.e., vehicle alone), or w positive control of CellRox induction (Figure 7).The structure of bituminarin C (3) was revealed to be slightly different from 1 and 2. The molecular formula C 25 H 28 O 6 that was deduced by the peak M-H − at m/z 423.1824 in the HR-ESIMS spectrum (negative mode) exhibited an additional oxygen atom with respect to 1 and 2. The 1 H NMR spectrum (Table 1) showed signals attributable to the pterocarpan core fused with a 3,3-dimethyl-2-hydroxy pyran ring, the same as 1 and 2, whereas differences were observed in the resonances due to the second prenyl chain linked at C-8.The spin system observed for the C-1 ′′ /C-5 ′′ fragment was constituted by a methylene resonating at δ H 2.78 (2H, m, overlapped signal, H 2 -1 ′′ ), which was coupled to a carbinolic methine at δ H 4.32 (1H, m, H-2 ′′ ) that was in turn long-range correlated with both a vinyl methyl at δ H 1.78 (3H, br s, H 3 -5 ′′ ) and an exomethylene at δ ( 1 H-1 H COSY, HSQC and HMBC) experiments allowed full proton and carbon assignment as reported in Table 1, also confirming the position of modified prenyl chains as reported in structure 3. The stereochemistry at C-6a and C-11a stereogenic centers was the same as 1 and 2, as was expected.The cis-junction was supported by the J 6a,11a value (7.0 Hz) and NOE interactions whereas the R,R absolute configurations at C-6a and C-11a were inferred by both the CD curve [31], and the negative specific rotation [α] D −71.5 (c 0.05, CHCl 3 ).The absolute configuration at C-2 ′ in bituminarin C (3) was determined by comparing the 1 H NMR pattern of H 2 -1 ′ of the corresponding acetyl derivative 3a with H 2 -1 ′ signals in compounds 1a and 2a exhibiting opposite C-2 ′ configuration (Figure 5).
The signal multiplicity of the methylene H 2 -1 ′ in compound 3a appeared similar with those of compound 2a, thus suggesting the same S configuration at C-2 ′ , whereas the configuration at C-2 ′′ remained unassigned.

Biological Activity: Antiproliferative Activity of Bitucarpin A (4) and Erybraedin C (5) on Neuroblastoma SH-SY5Y Cells
The biological properties of bitucarpin A (4) and erybraedin C (5) have been evaluated on neuroblastoma SH-SY5Y cells, that to the best of our knowledge have never been tested with pterocarpan compounds.First, with the aim at establishing the range of effective concentrations for the in vitro studies, a cell growth dose-response bioassay allowed us to determine the IC 50 of both bitucarpin A and erybraedin C. For this purpose, cell growth percentage was measured by a live-cell scan assay performed through the Zoom Incucyte system after 24 h of treatment with 4 and 5, both at log-fold concentrations establishing 100% of the cell growth from the untreated cells (Figure 6).This experiment aided in establishing IC 50 = 0.2048 µg/mL and IC 50 = 0.1976 µg/mL for bitucarpin A and erybraedin C, respectively.Successively, the effects on ROS production in SH-SY5Y cell line induced by bitucarpin A (4) and erybraedin C (5), both tested at 1 µg/mL and 0.1 µg/mL, were measured by CellRox assay in the same Zoom Incucyte live-cell scan system.Data were collected every two hours over 48 h of cell incubation by comparing with the time-matched cells incubated without pterocarpans (i.e., vehicle alone), or with LPS as positive control of CellRox induction (Figure 7).The signal multiplicity of the methylene H2-1′ in compound 3a appeared similar with those of compound 2a, thus suggesting the same S configuration at C-2′, whereas the configuration at C-2″ remained unassigned.

Biological Activity: Antiproliferative Activity of Bitucarpin A (4) and Erybraedin C (5) on Neuroblastoma SH-SY5Y Cells
The biological properties of bitucarpin A (4) and erybraedin C ( 5) have been evaluated on neuroblastoma SH-SY5Y cells, that to the best of our knowledge have never been tested with pterocarpan compounds.First, with the aim at establishing the range of effective concentrations for the in vitro studies, a cell growth dose-response bioassay allowed us to determine the IC50 of both bitucarpin A and erybraedin C. For this purpose, cell growth percentage was measured by a live-cell scan assay performed through the Zoom Incucyte system after 24 h of treatment with 4 and 5, both at log-fold concentrations establishing 100% of the cell growth from the untreated cells (Figure 6).This experiment aided in establishing IC50 = 0.2048 µg/mL and IC50 = 0.1976 µg/mL for bitucarpin A and erybraedin C, respectively.Successively, the effects on ROS production in SH-SY5Y cell line induced by bitucarpin A (4) and erybraedin C (5), both tested at 1 µg/mL and 0.1 µg/mL, were measured by CellRox assay in the same Zoom Incucyte live-cell scan system.Data were collected every two hours over 48 h of cell incubation by comparing with the time-matched cells incubated without pterocarpans (i.e., vehicle alone), or with LPS as positive control of CellRox induction (Figure 7).According to the literature [12,27,32], a time-and dose-dependent induction of the CellRox production was observed in SH-SY5H human neuroblastoma cell line treated with 4 and 5 at both concentrations (Figure 8).In particular, the analysis revealed a significant increase in the percentage of CellRox-positive cells after the first 10 h of incubation for both compounds at the higher tested dose.It is noteworthy that, after 32 h of incubation, bitucarpin A showed at 0.1 µg/mL a percentage of positive cells higher than that induced at 1 µg/mL (Figure 8).
With the aim of deeply investigating putative apoptotic induced effects by bitucarpin A (4) and erybraedin C (5), mitochondrial dynamics that have significance in the apoptotic signal transduction associated with cytochrome C release [33][34][35] were also analyzed.For this purpose, the "eccentricity index" variations induced by compounds 4 and 5 were measured.An eccentricity index value of 0.5 represents the morphological measure of the ideal circular shape typical of mitochondria in healthy cells [36,37] and deviation from this reference value indicates a cellular morphological hallmark predictive of apoptotic events [38,39].Mitochondrial dynamics were characterized, with single-organelle resolution, by live-cell microscopy associated with both time-lapse Zoom Incucyte image analysis software (version 20181.1.6628.28170)and supervised machine learning (Figure 9).Rhodamine-based staining dye MitoTracker was used in live cells treated with bitucarpin A and erybraedin C, over 48 h of incubation at 0.1 µg/mL and 1.0 µg/mL for both compounds.This analysis revealed a significant increase in the eccentricity mitochondrial index, highlighting an enhancement of the value close to 0.75 from the first 15 h of incubation for both bitucarpin A and erybraedin C at 1.0 µg/mL (Figure 10A1,B1).Additionally, putative apoptotic pathways involvedin cell damage induced by erybraedin C were further investigated in SH-SY5Y.Immunostaining of cytochrome C expression highlighted a morphological change of cytochrome C localization in cells treated with 1.0 µg/mL erybraedin C, observing a puncta-like distribution in the vehicle-treated cells vs. a widespread cytosolic localization in treated cells (Figure 11A-D and high magnification of boxed areas 1-4).These morphological changes were accompanied by both a significant reduction in the percentage of live cells (Figure 11E) and quantitative increase in cellular area expressing cytochrome C immunolabeling (Figure 11F).According to the literature [12,27,32], a time-and dose-dependent induction of th CellRox production was observed in SH-SY5H human neuroblastoma cell line treate with 4 and 5 at both concentrations (Figure 8).In particular, the analysis revealed a sign icant increase in the percentage of CellRox-positive cells after the first 10 h of incubatio for both compounds at the higher tested dose.It is noteworthy that, after 32 h of incub tion, bitucarpin A showed at 0.1 µg/mL a percentage of positive cells higher than th  With the aim of deeply investigating putative apoptotic induced effects by bitucarpin A (4) and erybraedin C (5), mitochondrial dynamics that have significance in the apoptotic signal transduction associated with cytochrome C release [33][34][35] were also analyzed.For this purpose, the "eccentricity index" variations induced by compounds 4 and 5 were measured.An eccentricity index value of 0.5 represents the morphological measure of the ideal circular shape typical of mitochondria in healthy cells [36,37] and deviation from this reference value indicates a cellular morphological hallmark predictive of apoptotic events [38,39].Mitochondrial dynamics were characterized, with single-organelle resolution, by live-cell microscopy associated with both time-lapse Zoom Incucyte image analysis software (version 20181.1.6628.28170)and supervised machine learning (Figure 9).Rhodamine-based staining dye MitoTracker was used in live cells treated with bitucarpin A and erybraedin C, over 48 h of incubation at 0.1 µg/mL and 1.0 µg/mL for both compounds.This analysis revealed a significant increase in the eccentricity mitochondrial index, highlighting an enhancement of the value close to 0.75 from the first 15 h of incubation for both bitucarpin A and erybraedin C at 1.0 µg/mL (Figure 10A1-B1).Additionally, putative apoptotic pathways involvedin cell damage induced by erybraedin C were further investigated in SH-SY5Y.Immunostaining of cytochrome C expression highlighted a morphological change of cytochrome C localization in cells treated with 1.0 µg/mL erybraedin C, observing a puncta-like distribution in the vehicle-treated cells vs. a widespread cytosolic localization in treated cells (Figure 11A-D and high magnification of boxed areas 1-4).These morphological changes were accompanied by both a significant reduction in the percentage of live cells (Figure 11E) and quantitative increase in cellular area expressing cytochrome C immunolabeling (Figure 11F).As under normal conditions cytochrome C resides in the mitochondria, exodus into the cytoplasm is considered as a marker of mitochondrial damage and apoptotic cell death by activation of key players, e.g., caspase-9 and the apoptotic effector caspases-3, 6, and 7 [33][34][35].To assess cell death by apoptosis, caspase-3 (Cas-3) and caspase-7 (Cas-7) expression was measured by immunolabeling SH-SY5Y cells treated with erybraedin C. A significant increase in the percentage of the immunolabeled cellular area was observed for both caspases (Figure 12).As under normal conditions cytochrome C resides in the mitochondria, exodus into the cytoplasm is considered as a marker of mitochondrial damage and apoptotic cell death by activation of key players, e.g., caspase-9 and the apoptotic effector caspases-3, 6, and 7 [33][34][35].To assess cell death by apoptosis, caspase-3 (Cas-3) and caspase-7 (Cas-7) expression was measured by immunolabeling SH-SY5Y cells treated with erybraedin C. A significant increase in the percentage of the immunolabeled cellular area was observed for both caspases (Figure 12).The induction of apoptosis in neuroblastoma cells was analysed by live cell scanning assay of caspase 3-7 reactivity (Figure 13).The effect of erybraedin C or bitucarpin A (1 µg/mL) was compared with rapamycin (0.01 µM) in order to confirm the induction of the pro-apoptotic cascade with a known anticancer drug.While in untreated (VEH) and in treated LPS the levels of capsase 3 and 7 remain low, in the treatment with erybraedin C and bitucarpin A, as well as in that with rapamycin, we observe an increase in cell death The induction of apoptosis in neuroblastoma cells was analysed by live cell scanning assay of caspase 3-7 reactivity (Figure 13).The effect of erybraedin C or bitucarpin A (1 µg/mL) was compared with rapamycin (0.01 µM) in order to confirm the induction of the pro-apoptotic cascade with a known anticancer drug.While in untreated (VEH) and in treated LPS the levels of capsase 3 and 7 remain low, in the treatment with erybraedin C and bitucarpin A, as well as in that with rapamycin, we observe an increase in cell death and a strong activation of caspases 3 and 7.The induction of apoptosis in neuroblastoma cells was analysed by live cell scannin assay of caspase 3-7 reactivity (Figure 13).The effect of erybraedin C or bitucarpin A µg/mL) was compared with rapamycin (0.01 µM) in order to confirm the induction of th pro-apoptotic cascade with a known anticancer drug.While in untreated (VEH) and treated LPS the levels of capsase 3 and 7 remain low, in the treatment with erybraedin and bitucarpin A, as well as in that with rapamycin, we observe an increase in cell dea and a strong activation of caspases 3 and 7.

Discussion and Conclusions
The chemical characterization of the pterocarpan fraction from the non-polar extract of B. bituminosa from Algeria resulted in the isolation and identification of a series of prenyl pterocarpans, including the new bituminarins A-C (1-3), as minor metabolites, and the main bitucarpin A (4) [4], erybraedin C (5) [18,19], and erybraedin D (6) [19,20] already reported from this species.The new compounds, bituminarins A-C (1-3), could be enzymatically derived from erybraedin C (5) by cyclization of C-4 prenyl group and further oxidation on the double bond to give the 2-hydroxy-3,3-dimethyldihydropyran ring.However, even though they were detected in the early crude extract fraction of the plant, a possible work-up origin could not be excluded for compounds 1-3.Prenyl substituents at C-4 and/or at C-8 of the pterocarpan framework are a distinctive structural feature of this group of metabolites that is scarcely distributed in nature and seems to be typical of Bituminaria plants [14].However, pterocarpans with similar prenyl residues, either linear or cyclized, have been recently reported also from Erythrina lysistemon [40] even though the substitution pattern of the pterocarpan nucleus was different from that described for Bituminaria metabolites.The presence of prenyl groups seems to enhance the bioactivities of the pterocarpan scaffold [27,41] as indicated by the broad spectrum of biological activities reported for prenylated pterocarpans [14].In this regard, the bioactivity evaluation of bitucarpin A (4) and erybraedin C (5) carried out on neuroblastoma SH-SY5Y cells adds further insight into the biomedical potential of these well-known bioactive compounds.Both bitucarpin A and erybraedin C were found to induce a significant reduction of the cell viability in neuroblastoma SH-SY5Y cells, revealing by time-lapse observation a time-dependent efficiency of both compounds to promote ROS production, mitochondria aggregation in a budding-like shape (i.e., eccentricity), and cytochrome C dislocation and spreading from mitochondria to cytoplasm [33,34].Pterocarpans constitute intermediate metabolites produced by specific redox reactions along the isoflavones biosynthesis [42].Erybraedin C, due to its phenolic hydroxyl and 4,8 prenyl substitutes, may exhibit a high chain-breaking antioxidant potential.Here, we report that erybraedin C induced a comparable dose-dependent cytotoxicity and dose-and time-dependent apoptosis in human glioblastoma cell line, unrelated to an apparent cell cycle checkpoint arrest.At the molecular level, this process may occur possibly by increasing the concentration of single-and double-stranded DNA breaks according to the previously reported effect of etoposide to stabilize a normally transient covalent DNA-topoisomerase II complex.This process triggers cell cycle arrest and activation of the biochemical cascade of terminal apoptotic events [43,44].Here, by using immunofluorescence staining, the induction of caspase-3 and caspase 7 was evidenced in concomitance with the pterocarpan-induced release of cytochrome C into the cytosol in cells undergoing apoptosis.These data are in accordance with previous studies reporting the antiproliferative effects of bitucarpin A and erybraedin C on human colon adenocarcinoma cells.In particular, erybraedin C has been proposed to induce DNA damage by stabilizing a transient covalent DNA-topoisomerase II complex [45], that is within diverse cellular stresses known to kill via the mitochondrial pathway of apoptosis [36].However, further studies on the mode of action underlying pterocarpan-induced apoptosome formation are required to better clarify their effects on the mitochondrial outer membrane permeabilization (MOMP)-induced apoptosis.

Plant Material
Bituminaria bituminosa was collected in the region of Fessdis (Batna), in June 2015.The plant was identified by Prof. Bachir Oudjehih of the Department of Agronomy of the Institute of Veterinary and Agronomic Science of the University of Batna 1.A voucher specimen (PB) was deposited at the Herbarium of the University of Batna-1, Batna, Algeria.

Preparation of MTPA Esters of 1
To a CH 2 Cl 2 solution (0.5 mL) of bituminarin A (1, 0.2 mg) a catalytic amount of DMPA and (R)-MTPA-Cl (3.5 µL) were added, and the mixture was stirred overnight at r.t.The reaction mixture was evaporated under vacuum to give a residue, which was purified by a pipette Pasteur silica gel column eluting with PE/DE (from 9:1 to 7:3) to give the corresponding (S)-MTPA ester (1b, 0.1 mg) of 1.Similarly, the (R)-MTPA ester (1c, 0.1 mg) of 1 was prepared following the same procedure using as reagent (S)-MTPA-Cl.
Cells were split and then plated after reaching 80% confluency, with densities from 4500 to 6000 cells/cm 2 at each passage.4.6.2.Immunofluorescence SH-SY5Y cells were fixed using 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4.Following fixation, samples were permeabilized 10 min with PB 0.3% Triton X 100 (Sigma-Aldrich, St. Louis, MI, USA) and finally incubated overnight with the primary antibodies Anti-Cytochrome C (1:100, mouse monoclonal Sc-13561 Santa Cruz Biotechnology, Paso Robles, CA, USA), anti-Caspase 3 (1:200, rabbit polyclonal AbCam, Cambridge, UK), and anti-Caspase 7 (1:100, Sc-56063, mouse monoclonal Santa Cruz Biotechnology, Paso Robles, CA, USA).After washing, the samples were then incubated for 2 h with a mixture of appropriate secondary antibodies Alexa Fluor 488 or 546 donkey anti-mouse or donkey anti-rabbit (Invitrogen) and the nuclei stained with DAPI (1 mg/mL; Sigma-Aldrich).Finally, the cells were mounted with an aqueous mounting medium (Aquatex, Merck, Darmstadt, Germany).Fluorescence-labeled cells were analyzed with a Leica DMI6000 fluorescence microscope equipped with an x-y-z motorized stage and cooled digital camera Leica K5 (Leica Microsystems, Buccinasco, Milan, Italy).Images were digitally acquired at the same magnification and processed for quantification of fluorescence by Leica LAS X software (Leica Application Suite X 3.7.4.23463).The optimal focus was chosen for each fluorescence channel of the z-stacked images and merged to obtain the best multichannel image for each well analyzed to measure the percentage of immunolabeled area/well for each immunostaining.All the measurements were performed in quadruplicate.Statistical and quantification analyses were conducted by an observer blinded to the experimental design using Fiji, an image-processing package of ImageJ software (version 2.14.0) developed by the National Institutes of Health, USA.The LIVE/DEAD ® Viability/Cytotoxicity two-color assay (Invitrogen) was applied to the cells before and after erybraedin C 1 µg/mL treatment to determine the percentage of cell viability by fluorescence microscopy.

Incucyte Live Cell Scanning
To observe the temporal development of the oxidative stress, and mitochondrial morphology of SH-SY5Y cells in response to bitucarpin A and erybraedin C treatment, a live action imaging assay was performed using the Zoom Incucyte scanner (Sartorius).Human neuroblastoma SH-SY5Y cells were plated in 24× multi wells with a density of 10,000/15,000 cells per well.The multi wells were inserted into the scanner placed in the incubator in order to preserve the ideal environmental conditions and were scanned at progressive time points by covering a framework of 48 h.Each well was scanned by keeping images from 16 different areas/well; that is the maximum number of images acquired for the best proxy-live representation for wells.Each well was scanned every 30 min; that is the time required for cooling the mechanical components of the scanner.The image acquisition was performed with 20× magnification objective in phase contrast and red fluorescence.All images were then analyzed using the integrated Zoom Incucyte software (version 20181.1.6628.28170)after appropriate calibration of analysis.Quantitative analysis provided the time-based trend of the percentage of cells labelled with CellRox or Mitotracker which were identified in the scanned areas for each well within the time lapse scanning.
The SH-SY5Y cells were seeded 4000 cells × well in 24 wells plates to be tested by growth inhibition assay, identifying the IC 50 value of growth inhibition for bitucarpin A and erybraedin C.

Figure 6 .
Figure 6.SH-SY5Y cell growth assay.IC50 values are measured following 24 h of cell treatment with bitucarpin A or erybraedin C at dose ranging from 0.01 µg/mL to 5 µg/mL in comparison to untreated, control (CTRL) cells.

Figure 6 .
Figure 6.SH-SY5Y cell growth assay.IC 50 values are measured following 24 h of cell treatment with bitucarpin A or erybraedin C at dose ranging from 0.01 µg/mL to 5 µg/mL in comparison to untreated, control (CTRL) cells.

Figure 10 .
Figure 10.Time course analysis (48 h) of Mitotracker response to oxidative stress in SH-SY5Y cell line.(A1) Line-plot graph representing the time-course Mitotracker reactivity of cells treated with or without bitucarpin A (0.1 and 1 µg/mL) in comparison with LPS treatment.(B1) Line-plot graph of Mitotracker reactivity of cells treated with or without erybraedin C (0.1 and 1 µg/mL) in comparison with LPS treatment.(A2) Bar-plot quantitative analysis (t0, t24 and t48) of mitochondrial eccentricity changes induced by bitucarpin A treatment (at 0.1 µg/mL and 1 µg/mL) vs. control.(B2) Barplot quantitative analysis (t0, t24 and t48) of mitochondrial eccentricity changes induced by erybraedin C treatment (at 0.1 µg/mL and 1 µg/mL) vs. control.The y axis is eccentricity defined in an ellipse as the ratio of the distance between its center and either of its two foci (from 1 for circular shapes to 0 for straight lines); the x axis is the time in hours.Two-way ANOVA with Tukey post-hoc test was used to assess statistical significance (* p < 0.05, ** p < 0.01, **** p < 0.0001; error bars represent mean ± SEM; (n = 48).

Figure 10 .
Figure 10.Time course analysis (48 h) of Mitotracker response to oxidative stress in SH-SY5Y cell line.(A1) Line-plot graph representing the time-course Mitotracker reactivity of cells treated with or without bitucarpin A (0.1 and 1 µg/mL) in comparison with LPS treatment.(B1) Line-plot graph of Mitotracker reactivity of cells treated with or without erybraedin C (0.1 and 1 µg/mL) in comparison with LPS treatment.(A2)Bar-plot quantitative analysis (t0, t24 and t48) of mitochondrial eccentricity changes induced by bitucarpin A treatment (at 0.1 µg/mL and 1 µg/mL) vs. control.(B2) Bar-plot quantitative analysis (t0, t24 and t48) of mitochondrial eccentricity changes induced by erybraedin C treatment (at 0.1 µg/mL and 1 µg/mL) vs. control.The y axis is eccentricity defined in an ellipse as the ratio of the distance between its center and either of its two foci (from 1 for circular shapes to 0 for

Figure 11 .
Figure 11.Erybraedin C affects Mitotracker and cytochrome C expression in SH-SY5Y cells.(A-D) Images of triple Mitotracker (Red)/cytochrome C (Green)/DAPI (Blue) labeled vehicle-treated cells (A merge, and B single cytochrome C immunolabeling) in comparison to 24 h treatment with erybraedin C (1 µg/mL) (C merge, and D single cytochrome C immunolabeling).Dotted boxes inside each picture represent the respective field acquired with high magnification showing cellular details of SH-SY5Y cells in 1-4.Scale bar = 100 µm in A-D, and 10 µm in 1-4.(E) Bar-plot analysis of the mean of percentage of live cells before and after erybraedin C treatment as revealed by live and dead assay.(F) Bar-plot analysis of the mean of percentage of the cytochrome C-positive immunolabeled

Figure 11 .
Figure 11.Erybraedin C affects Mitotracker and cytochrome C expression in SH-SY5Y cells.(A-D) Images of triple Mitotracker (Red)/cytochrome C (Green)/DAPI (Blue) labeled vehicle-treated cells (A merge, and B single cytochrome C immunolabeling) in comparison to 24 h treatment with erybraedin C (1 µg/mL) (C merge, and D single cytochrome C immunolabeling).Dotted boxes inside each picture represent the respective field acquired with high magnification showing cellular details of SH-SY5Y cells in 1-4.Scale bar = 100 µm in A-D, and 10 µm in 1-4.(E) Bar-plot analysis of the mean of percentage of live cells before and after erybraedin C treatment as revealed by live and dead assay.(F) Bar-plot analysis of the mean of percentage of the cytochrome C-positive immunolabeled area/cell.Two-way ANOVA with Tukey post-hoc test was used to assess statistical significance (* p < 0.05; ** p < 0.01; error bars represent mean ± SEM).

4. 6
.4.Reagents The carbocyanine-based MitoTracker™ Deep and the Red CellROX™ Deep Red were exploited as live-cell fluorescent probes having the advantages of not interfering with cellular vitality during the analysis of the cell responses to the oxidative stress.The carbocyaninebased MitoTracker™ Deep Red (Thermo Fisher Scientific Inc. Waltham, MA, USA) has active